1. Field of the Invention
The field of this invention is antisense oligodeoxynucleotides and pharmaceuticals based on them.
2. Description of Related Art
The ACHE gene encoding the acetylcholine hydrolyzing enzyme acetylcholinesterase (AChE, EC 3.1.1.7) is expressed in muscle, nerve, hematopoietic cells, embryonic tissue and germ cells. ACHE maps to chromosome 7q22 and encodes the primary enzyme, acetylcholinesterase (AChE, E.C. 3.1.1.7), which terminates neurotransmission at synapses and neuromuscular junctions.
The text Human Cholinesterases and Anticholinesterases by Soreq and Zakut (Academic Press, Inc., 1993) provides a summation of the biochemical and biological background as well as the molecular biology of human cholinesterase genes. The text in its entirety is incorporated herein by reference. Further, the text Transgenic Xenopus by Seidman and Soreq (Humana Press, 1996) provides a summation of the development of the Xenopus transgenic animal model. The text in its entirety is incorporated herein by reference. Articles by Beeri et al, 1995; Karpel et al, 1996; and the review articles by Lev-Lehman et al, 1997 and Grifman et al, 1995, 1997 provide further information on the development of antisense ACHE oligomers, the parameters for choosing sequences and testing for efficacy and are incorporated herein by reference.
Briefly, AChE includes the peptide motif S/T-P-X-Z, which makes it a potential substrate for phosphorylation by cdc2 kinases, the general controllers of the cell cycle. Most other substrates of cdc2 kinases perform biological functions necessary for cell cycle-related processes. Thus, interference with either CHE or cdc2 transcription processes may be expected to divert and/or arrest cell division, and controlling these processes can be useful for several, medically important procedures.
Biochemical and histochemical analyses indicate that AChE is expressed, in high levels, in various fetal tissues of multiple eukaryotic species where cholinesterases (ChEs) are coordinately regulated with respect to cell proliferation and differentiation. The specific role to be attributed to ChEs in embryonic development may hence be related with cell division, so that their biological function(s) in these tissues are tentatively implicated in the control of organogenesis.
In addition to its presence in the membranes of mature erythrocytes, AChE is also intensively produced in developing blood cells in vivo and in vitro and its activity serves as an acceptable marker for developing mouse megakaryocytes. Furthermore, administration of acetylcholine analogues as well as cholinesterase inhibitors has been shown to induce megakaryocytopoiesis and increased platelet counts in the mouse, implicating this enzyme in the commitment and development of these hematopoietic cells.
A major hydrophilic form of AChE with the potential to be "tailed" by non-catalytic subunits is expressed in central nervous system and muscle whereas a hydrophobic, phosphoinositide (PI)-linked form of the enzyme is found in erythrocytes. Alternative exons encoding the C-terminal peptide in AChE were shown to provide the molecular origins for the amphiphilic (PI)-linked and the hydrophilic "tailed" form of AChE in Torpedo electric organ. The existence of corresponding alternative exons and homologous enzyme forms in mammals suggested that a similar mechanism may provide for the molecular polymorphism of human AChE. cDNAs reported to date from mammalian brain and muscle encode the hydrophilic AChE form.
More specifically, three alternative AChE-encoding mRNAs have been described in mammals (FIG. 11). The dominant central nervous system and muscle AChE (AChE-T) found in the neuromuscular junction (NMJ) is encoded by a mRNA carrying exon E1 and the invariant coding exons E2, E3, and E4 spliced to alternative exon E6 [Li et al., 1991; Ben Aziz-Aloya et al., 1993]. AChEmRNA bearing exons E1-4 and alternative exon E5 encodes the glycolipid phosphatidylinositol (GPT)-linked form of AChE characteristic of vertebrate erythrocytes (AChE-H;) [Li et al., 1993; Legay et al., 1993a]. An additional readthrough mRNA species retaining the intronic sequence I4 located immediately 3' to exon E4 was reported in rodent bone marrow and erythroleukemic cells [Li et al., 1993; Legay et al., 1993a] and in various tumor cells lines of human origin [Karpel et al., 1994].
AChE is the major cholinesterase (ChE) in nervous system cells. Since acetylcholine is produced mostly in the CNS, changes in AChE should be coupled to mental state.
The cholinergic theory of Alzheimer's disease [Coyle, et al, 1983; Slotkin et al., 1994] suggests that the selective loss of cholinergic neurons in Alzheimer's disease results in a relative deficit of acetylcholine in specific regions of the brain that mediate learning and memory functions and require acetylcholine to do so. The primary approach to treating Alzheimer's disease has therefore aimed to augment the cholinergic system. Reduced levels of acetylcholine in the brains of Alzheimer's patients leaves a relative excess of acetylcholinesterase, the enzyme responsible for terminating nerve impulses during normal brain activity by disposing of used acetylcholine (Soreq and Zakut, 1993). A relative excess of acetylcholinesterase accentuates the growing cholinergic deficit by further reducing the availability of acetylcholine. The most successful strategy to date for reinforcing cholinergic neurotransmission in Alzheimer's patients is pharmacological inhibition of acetylcholinesterase. Indeed, the only currently approved drugs for Alzheimer's disease are potent acetylcholinesterase inhibitors (Winker, 1994), i.e. drugs that suppress the catalytic activity of the acetylcholine hydrolyzing enzyme acetylcholinesterase (acetylcholine acetyl hydrolase, EC 3.1.1.7, AChE) [Knapp et al., 1994]. This provides augmentation of cholinergic neurotransmission, which is impaired in such patients due to the selective loss of cholinergic neurons. However, such inhibitors do not reduce the amount of AChE protein, and there are recent reports of actions of AChE, unrelated to its catalytic activity, in process extension [Small et al., 1995, Layer et al.; 1995, Jones et al., 1995; Darboux et al., 1996; Sternfeld et al., 1997] and amyloid fibril formation [Inestrosa et al., 1996].
Tacrine, the first and most well-characterized acetylcholinesterase inhibitor used for treating Alzheimer's disease offers limited palliative relief to 30-50% of mild-moderately affected Alzheimer's patients for up to 6 months [Winker, 1994; Krapp et al., 1994]. The positive, albeit partial, success of Tacrine attests to the utility of the cholinergic theory and the potential value of improved anticholinesterase treatment for Alzheimer's disease. Another approved drug, E-2020 (Aricept) has been reported to follow the same mode of action as Tacrine but at lower doses [Rogers et al., 1996]. A number of other compounds are under development for inhibition of acetylcholinesterase [Johansson and Nordberg, 1993]. All of these are aimed at blocking the fully folded protein from degrading acetylcholine.
The positive, albeit partial, success of Tacrine attests to the potential value of improved anticholinesterase treatment of Alzheimer's Disease. However, anti-acetylcholinesterase therapies for Alzheimer's Disease require high doses of drug and produce side-effects associated with systemic cholinergic toxicity. Tacrine, for example, has been associated with liver damage and blood disorders in some patients [Johansson and Nordberg, 1993]. Further, current ACHE inhibitors interact non-specifically with the AChE-homologous serum protein butyrylcholinesterase and stimulate regulatory feedback pathways leading to enhanced expression of AchE.
Although newer inhibitors such as E-2020 having greater specificity for acetylcholinesterase provide for lower doses [Rogers et al., 1996], they are not likely to completely overcome the problem of cross-reactivity with butyrylcholinesterase, given the high degree of similarity between the two proteins [Loewenstein-Lichtenstein et al., 1996]. Moreover, liver function, red blood cell counts, and natural variations in the genes encoding both acetylcholinesterase and butyrylcholinesterase will determine both the quantity and quality of the drug scavenging potential among individual patients. Several mutations in the butyrylcholinesterase gene already have been suggested to create a genetic predisposition for adverse responses to anti-cholinesterases [Loewenstein-Lichtenstein et al., 1995]. This implies that even in the best case scenario for acetylcholinesterase inhibitor-based therapies, various elements must be considered in designing individualized dosage regimens on a patient-by-patient basis.
Myasthenia gravis (MG) is an autoimmune disease in which antibodies directed against the nicotinic acetylcholine receptor (AChR) at the motor end plate of the neuromuscular junction (NMJ) by binding to the ACHR impair neuromuscular communication either directly or through NMR degradation. Therapeutic strategies for treating MG now include anticholinesterase drugs, immunosuppressive drugs, thymectomy and plasmapheresis [see Myasthenia Gravis And Related Disorders, Annals of the New York Academy of Sciences, volumes 681 (1993), 505 (1987), 377 (1981), 274 (1976) and 135 (1966) for an overview of the progress and development of the understanding of MG disease etiology and therapeutic strategies as well as the 1998 volume (in press)].
AChE is accumulated at neuromuscular junctions (Salpeter 1967) where it serves a vital function in modulating cholinergic neurotransmission (reviewed by Soreq and Zakut, 1993). The molecular mechanisms by which AChE and other synaptic proteins are targeted to the NMJ are poorly understood. Compartmentalized transcription and translation in and around the junctional nuclei probably contribute to the NMJ localization of AChE (Jasmin et al., 1993). Therefore, anticholinesterase therapy is utilized with almost all patients in order to reduce AChE and thereby increase the halflife of acetylcholine thereby potentiating neuromuscular transmission. This therapy is often used in concert with immunosuppressive therapy (generally steroids). It is the general goal to remove the patient from immunosuppressive therapy due to its side effects.
Pyridostigmine (Mestinon.RTM.) remains the drug of choice in treating myasthenics due to its effectiveness and tolerance by patients. Ambenonium (Mytelase.RTM.) is used for those MG patients who cannot tolerate pyridostigmine. However, patients must be involved in the determination of dosage of the drug since dosage will often need to be adjusted over any twenty-four hour period. Variables such as menstrual cycle, infections and emotional stress affect dosage and patients must be trained to modify their dosage to take these and other factors into consideration. Overdosage of pyridostigmine can lead to cholinergic crisis and even with good patient education such overdoses occur. Additionally, as discussed herein above genetic predispositions to predisposition for adverse responses to anti-cholinesterases [Loewenstein-Lichtenstein et al., 1995] must be considered.
Cholinergic crisis due to anticholinesterase drug overdose is characterized by increasing muscle weakness which if involving the respiratory muscles can lead to a myasthenic crisis and death. Myasthenic crisis due to increase in severity of disease can also present with the same symptoms. Distinguishing between the two is extremely important since treatment for a cholinergic crisis is termination of anticholinesterase drugs while the non-drug associated myasthenic crisis would indicate an increase in anti-cholinergic drug dosage. Therefore finding alternatives to anticholinesterase drug therapy in MG would be useful.
These considerations indicate the need to develop a new generation of anti-acetylcholinesterase drugs displaying increased target specificity, improved efficacy and reduced side effects.
Breakthroughs in molecular biology and the human genome project have opened previously unforeseen possibilities for targeted intervention with mammalian gene expression. These include permanent approaches such as transgenic overexpression or recombinant disruption of specific genes as well as novel approaches for transient suppression of gene function. Short synthetic antisense (AS) oligodeoxynucleotides (AS-ODN) designed to hybridize with specific sequences within a targeted mRNA belong to the latter class.
Many excellent reviews have covered the main aspects of antisense technology and its enormous therapeutic potential. The literature naturally progressed from chemical [Crooke, 1995] into cellular [Wagner, 1994] and therapeutic [Hanania, et al, 1995; Scanlon, et al, 1995] aspects of this rapidly developing technology. Within a relatively short time, ample information has accumulated about the in vitro use of AS-ODN in cultured primary cells and cell lines as well as for in vivo administration of such ODNs for suppressing specific processes and changing body functions in a transient manner. This wealth of accumulated experience now offers a novel way to analyze the antisense approach, namely, to compare its in vitro uses with its in vivo ones [Lev-Lehman et al, 1997]. Further, enough experience is now available in vitro and in vivo in animal models as shown in the Examples of the present application to predict human efficacy.
AS intervention in the expression of specific genes can be achieved by the use of synthetic AS-ODNs [for recent reports see Lefebvre-d'Hellencourt et al, 1995; Agrawal, 1996; Lev-Lehman et al, 1997]. AS-ODNs are short sequences of DNA (averaging 15-25 mer) designed to complement a target mRNA of interest and form an RNA:ODN duplex. This duplex formation can prevent processing, splicing, transport or translation of the relevant mRNA. Moreover, certain AS-ODNs can elicit cellular RNase H activity when hybridized with their target mRNA, resulting in mRNA degradation [Calabretta et al, 1996]. In that case, RNase H will cleave the RNA component of the duplex and can potentially release the AS-ODN to further hybridize with additional molecules of the target RNA. An additional mode of action results from the interaction of AS-ODNs with genomic DNA to form a triple helix which may be transcriptionally inactive. See FIG. 1 for a schematic representation of the modes of action of AS-ODN.
Phosphorothioate antisense oligonucleotides do not show significant toxicity and exhibit sufficient pharmacodynamic half-lives in animals [Agarwal et al., 1991, 1996]. Antisense induced loss-of-function phenotypes related with cellular development were shown for the glial fibrillary acidic protein (GFAP), implicated in astrocyte growth within astrocyte-neuron cocultures [Winstein et al., 1991], for the myelin-associated glycoprotein in Schwann cells, responsible for formation of the compact myelin sheath formation surrounding these cell [Owens and Bunge, 1991], for the microtubule-associated tau proteins implicated with the polarity of hippocampal neurons and their axon formation [Caceres and Kosik, 1990], for the .beta..sub.1 -integrin, important for neuronal migration along radial glial cells, and for the establishment of tectal plate formation in chick [Galileo et al., 1991] and for the N-myc protein, responsible for the maintenance of cellular heterogeneity in neuroectodermal cultures (ephithelial vs. neuroblastic cells, which differ in their colony forming abilities, tumorigenicity and adherence) [Rosolen et al., 1990; Whitesell et al, 1991]. Antisense oligonucleotide inhibition of basic fibroblast growth factor (bFgF), having mitogenic and angiogenic properties, suppressed 80% of growth in glioma cells [Morrison, 1991] in a saturable and specific manner. The antisense oligonucleotides were targeted against the initiation and splice sites in bFgFmRNA, they reduced activity of the resulting protein and sense oligomers remained inactive. In soft-agar cultures, antisense oligonucleotides reduced the size of glial colonies and induced appearance of larger cells within them [Morrison, 1992]. Being hydrophobic, antisense oligonucleotides interact well with phospholipid membranes [Akhter et al., 1991]. Following their interaction with the cellular plasma membrane, they are actively transported into living cells [Loke et al., 1989], in a saturable mechanism predicted to involve specific receptors [Yakubov et al., 1989].